Stem cells (SC) are uniform undifferentiated cells having the property of constant regeneration and the unique ability of turning into any other cell type by cleavage and differentiation. By said potential, SC are a renewable source of human tissue. Thus, SC be-came an important object of medical research for various applications, such as gene therapy, organ transplantation, diabetes, and plastic surgery.
SC may be divided into two groups, i.e. embryonic and adult (EC). Embryonic stem cells play a key role in the first development phase of an organism. They are able to endlessly cleave and to develop every necessary type of tissue. Thus, they are able to form from a single cell a whole body, either the plant or the animal form which they originate. Due to this ability they are also called pluripotent cells. Unfortunately, for human beings this ability is restricted to the embryonal phase. In later phases of life of a subject EC are no longer present.
The second type of stem cells are adult stem cells. So far, these cells could be identified in many full-grown tissues and organs, such as bone marrow, pancreas, spine, brain, central nervous system, peripheral blood, dental pulp, blood vessel, skeletal muscles, cornea, retina, liver, cord blood, heart, epithelium of the intestinal tract, and dermis.
Compared with EC adult SC derived from such tissues have only a limited choice of differentiation. Some of them can only differentiate into one single tissue type mostly the one which is surrounding them. Thus, they are called unipotent SC. Other SC can differentiate into various tissue types and therefore are called multipotent.
Both types of adult SC are promising for medical applications since they are more easily accessible and their recovery is ethically less problematical than of ES. A survey on SC and their possibilities may be gleaned in Lemoni et al., 2005, Stem Cell Plasticity: Time for a Reappraisal, Hematologica/The Hematology Journal, 90 (3), 360 to 381.
The skin of mammals is a multilamina system which is continuously revolving. The part which is constantly in contact with the outside world is called epidermis. The major task of this specialized Tissue is to protect the body against dehydration, lesions and infections. It is composed four different laminas which are all formed by a single cell type, the so-called cerationocytes. Whereas this cell type is not much differentiated, is nevertheless has its origin in specialized skin stem cells. They are located in the lowermost lamina of the epidermis, the basal lamina.
In several papers successful isolation of such skin stem cells is reported. It even could be demonstrated that skin stem cells may be found in lower tissues of the skin, i.e. in the so-called hair follicle bulge. Contrary to the SC in the basal lamina these SC are multipotent, i.e. they are able to differentiate into every tissue type of the skin. A survey may be gleaned in: Roh et al., 2006, Cutaneous Stem Cells and Wound Healing, Pediatric Research, 59 (4), Pt 2, 100 to 103R; Morasso et al., 2005, Epidermal Stem Cells; The Cradle of Epidermal Determination, Differentiation and Wound Healing, Biol. Cell., 97, 173 to 183; and Alonso et al., 2003, Colloquium: Stem Cells of the Skin Epithelium, PNAS, 100, Suppl. 1, 11830 to 11835.
Skin stem cells are crucial in the wound healing and the regeneration of skin and hair. However, the capacity of these abilities may be disturbed by genetic problems, environmental influences and the aging process. Thus, protection of these SC is extremely important. Therefore, as will be explained below more in detail, it was an object of the present invention to develop a plant extract able to protect and stimulate these SC in cosmetic preparations.
Plant extracts and the use of parts of plants, such as leaves, fruits, flowers, stems, bark, inflorescences and roots for cosmetic and medical Applications are known since ancient times. Products derived therefrom may be, e.g., essential oils, fibers, starch, flavors, coloring matters, antibiotics, proteins, phenols, acids or fats. The use of plants or plant extracts in cosmetics is rampant. There are a great many of different uses, such as humidification, brighteners, tanning lotions, make-ups, sun filters, scavengers, antioxidants, immunity stimulation, detergents, preserving agents or thickening agents.
Examples of recently found uses are described in: KR20040091178, KR20040059007, US20062400129, WO2006099930, WO2006086707, US2006153792, JP2006151934, WO2006068777, WO2006053761, WO2006008418, LV13345, UA73556, CN1679498, and many others.
The spectrum of useful plants and plant component is wide and comprises e.g. algae, succulents, berries, carnivorous plants, herbs, cereals and trees. Usual well known examples of plants, however not limited to them, are: Spirulina algae, aloe vera, calendula, ginkgo, ginseng, iris, valerian, sage, lavender, thyme, peppermint, Saint-John's-wort, citrons, peach, guava, avocado, wheat, and oat.
However there are restrictions with respect to the use of plants or plant components. i.e.:                The availability may be restricted, e.g. by the seasons, limited storage capacities, protection of species, problems in cultivation, or bad harvests.        The quality is not unchanging, e.g. due to seasonal variations, different cultivation methods, geographic differences, different suppliers, clones, pollution of the environment, or physical status.        
These facts often make the use of plants in cosmetic applications impossible.
Therefore, the utilization of methods of plant cell culture techniques may help to solve such problems. Said utilization comprises techniques which allow, when observing certain known process steps, to obtain uniform dedifferentiated cells, showing the following advantages as compared with cultivated whole plants:                Independency from seasons;        Continuous production:        Freedom from pollution of environment and other impurities;        With respect to quantity and quality manageable and reproducible production of metabolites;        Protection of rare or limited plant reserves;        No limitation of market availability.        
Examples for the use of plant cell cultures of various species, their cultivation and their use in cosmetic preparations may e.g. be found in: EP1244464, FR2837385, US2006021084, WO2005108596, WO2005070066.
The basic principle of cultivation of such dedifferentiated plant cells utilizes the biological fact, that every plant cell has the ability to build up the whole plant which the cell stems from. This ability is called totipotency and is comparable with the pluripotency of animal ES. Therefore, it may be accepted that dedifferentiated plant cells do have a positive influence on protection and activation of skin stem cells.
In order to achieve this effect various dedifferentiated plant cells can be used. However, further supplemental useful effects can be achieved using these plant extract as well. An investigation within several plant groups showed that apples and fruits belonging to the subfamily Maloideae of the family of Rosaceae are much promising. A known exponent of this family is the cultivated apple tree (Malus domestica). Apples have a long tradition in cosmetic applications. Originally, they were applied in the form of masks of pressed pulp or peelings which provided moisture and tautness of the skin. Another application is the use of apple aromas and extracts in all kinds of cosmetic preparation, such as e.g. shampoos, lotions, soaps, bath essences or toothpastes.
The main ingredients of apples are various sugars, vitamins, acids, oils, waxes and polyphenols. Recently published studies could prove that especially the overall polyphenols in apple extracts or apple juices rich in polythenols can be useful in preventing and combating colon cancer (Eberhart et al., 2000, Antioxidant Activity of Fresh Apples, Nature, 405, 903 to 904; Liu et al., 2003, Antiproliferative Activity of Apples is not due to Phenolic-induced Hydrogen Peroxide Formation, J. Agric. Food Chem., 51, 1718 to 1723; Kern et al., 2005, Inhibitors of the Epidermal Growth Factor Receptor in Apple Juice Extract, Mol. Nutr. Food Res., 49, 317 to 328). Thereby, to a certain extent, the juices rich in polyphenols had an influence on the Wnt-pathway. This pathway is a cytobiological signaling cascade in which β-catenin is the main protein. Under normal circumstances, this protein is present in the cell on a constant level. If this level is disturbed, as in a cancer cell, the β-catenin level rises, and the β-catenin is transported into the cell nucleus, where it initiates the transcription of genes which causes an uncontrolled cleavage. Kern et al. (2006, Modulation of Key Elements of the Wnt-Pathway by Apple Polyphenols, J. Agric. Food. Chem., 54, 7041 to 7046) could show that the level of intracellular β-catenin in colon cancer cells cultivated in vitro was reduced by administration of apple juice.
Furthermore, it was found that apple show a large antioxidative activity and can increase the antioxidative capacity in blood (Rezk et al., 2002, The Antioxidant Activity of Phloretin: The Disclosure of a new Antioxidant Pharmacophore in Flavonoids, Biochem. Biophys. Res. Commun., 295, 9 to 13; Lee et al., 2003, Major Phenolics in Apple and their Contribution to the Total Antioxidant Capacity, J. Agric. Food Chem., 51, 6516 to 6520; Vrohovsek et al., 2004, Quantitation of Polyphenols in Different Apple Varieties, J. Agric. Food Chem., 52, 6532 to 6538; Lotito et al., 2004, Relevance of Apple Polyphenols as Antioxidants in Human Plasma: Contrasting in-vitro and in-vivo Effects, Free Rad. Biol. Med., 36, 201 to 211; Bitsch et al., 2000, Bioavailability of Antioxidative Compounds from Brettacher Apple Juice in Humans, Food Sci. Emerg. Technol., 1, 245 to 249). For this reason, apples are very interesting for establishing a dedifferentiated cell culture and its subsequent use in cosmetic preparations.
Dedifferentiated plant cells have a complex matrix of constituents of salts, acids, polyphenols, sugars, fats, proteins and other components. In addition to known components there is an unknown fraction of components which possibly is very valuable for cosmetic applications. It is known that raw plant extracts often show a better effect than identified and isolated individual components. Therefore, it is reasonable to use the entire cell lysate for application.
In order to obtain such total fraction of all ingredients special techniques are required since part of them are water-soluble whereas another part is fat-soluble. It was proposed to process plant cell culture preparations by means of lyophilization (e.g. WO2005072697, US20050265953). Thereafter, these lyophilized cells were pulverized and used in topic preparations.
Since transport of materials through the skin barrier is very limited, the technique of producing liposomes for many cosmetic applications was developed (e.g. KR20050091162, KR920005639B, GB2415375, WO2004067012, EP1498420, US2002160064, AU2388099). Application of this technique allows a better penetration of substances into the lower skin laminas. Also, a further advantage of liposomes is the encapsulation of fat-soluble ingredients in the membrane and thus their dispersion in aqueous phases.
There are various methods of liposome production. Main steps of their production comprise dissolving a phospholipid mixture in a suitable solvent (e.g. glycerol or alcohol), intermixing the dissolved lipids with an aqueous phase, applying energy (e.g. by stirring, shaking, pressure or heat) for forming the liposomes. As said above, the form of energy can by pressure. Formation of liposomes by means of high pressure homogenization is a known technique. Examples for pharmaceutical or cosmetic preparations may be found e.g. in WO9949716, NZ502840 or EP0782847. Interestingly the same technique can be used for solubilizing cells and obtaining their lysate (e.g. DE19918619). Therefore, it is possible to solubilize plant cells of suspension cultures and at the same time to extract the oil- and water-soluble agents into empty liposomes. Thereby, stability of the agents and their transportation into the skin can be improved.
From the above mentioned publication WO 2005/072697 A1 it is known to use lyophilizates of dedifferentiated plant cells for depigmenting the skin. This technique calls for the use of lyophilizates of dedifferentiated plant cells, in particular of cells of halophile plants. A use for stimulation and protection of skin stem cells is not envisaged.
From the publication EP 1,174,120 A1 it is known to use extracts, in particular of lyophilizates, of dedifferentiated cells of plants of the family Iridaceae (Iris-family) for stimulating immunity. Other plants or uses are not proposed.
The publication EP 1,064,932 A1 proposes the use of extracts of dedifferentiated plant cells in deodorants. Other uses are not disclosed.
The publication WO 03/077881 A discloses the use of lysates of metabolites of dedifferentiated cells of vine, which were obtained by means of a complicated method, for the preparation of cosmetics. This technique calls for the use of lyophilizates. A use for stimulation and protection of skin stem cells is not envisaged. Furthermore, other species of plants are not disclosed.
Furthermore, the publication WO 01/47538 A1 discloses the use of extracts of dedifferentiated cells of plants of the genus Leontopodium (Edelweiss) as UV filter. Other uses or other plants are not proposed.